The present invention relates to the identification of proteins useful for improving tolerance to water deficit in plants, to nucleic acid sequences encoding said proteins, and to their use for increasing the tolerance of plants to water deficit.
“Water deficit” relates to a situation where a plant has insufficient water for optimal functioning of its physiological processes.
Water deficit is one of the most important abiotic stresses affecting plants, and is one of the main factors responsible for yield loss in crops. Water deficit can severely affect plant yield, growth and reproduction. Therefore, it is important to identify genes that have the capacity to improve plant tolerance to water deficit.
The plant hormone abscisic acid (ABA) is renowned for being a stress hormone. Its levels rise in response to abiotic stress, in particular upon water deficit, and it has been shown to be necessary for the induction of a variety of adaptive responses from stomatal closure to osmotic adjustment (NAMBARA and MARION-POLL, Annu Rev Plant Biol, 56, 165-85, 2005).
ABA is a sesquiterpenoid (C15) that is synthesized in plants via an indirect pathway using carotenoid precursors. In plants carotenoids are essential components of the photosynthetic apparatus and are the red, orange and yellow pigments found in many flowers and fruit. Early ABA biosynthesis reactions occur, therefore, in plastids. The direct precursors of ABA are the xanthophylls zeaxanthin, violaxanthin and neoxanthin with the first ABA biosynthesis specific reaction being the oxidative cleavage of 9-cis-epoxycarotenoids (FIG. 1). The cleavage product xanthoxin is then transited to the cytoplasm where it undergoes conversion to abscisic aldehyde followed by oxidation to yield ABA. The biosynthetic pathway of ABA is schematized on FIG. 1.
Genes encoding the enzymes for most of the steps of the ABA biosynthesis pathway have been cloned or their function confirmed using ABA-deficient mutants. The zeaxanthin epoxidase (ZEP) gene was first cloned from a Nicotiana plumbaginifolia insertion mutant and is defective in the Arabidopsis aba1 mutant (MARIN et al., Embo J, 15, 2331-42, 1996). The ZEP enzyme catalyses the conversion of zeaxanthin to violaxanthin via antheraxanthin in two successive epoxidation reactions (FIG. 1). The maize VP14 mutant is mutated in a 9-cis-epoxycarotenoid dioxygenase (NCED) gene required for oxidative cleavage (SCHWARTZ et al., Science, 276, 1872-4, 1997; TAN et al., Proc Natl Acad Sci USA, 94, 12235-40, 1997) and in vitro activity assays suggested that either violaxanthin or neoxanthin cis-isomers could be substrates. A short chain dehydrogenase/reductase (SDR) catalyses the oxidation of xanthoxin to abscisic aldehyde (FIG. 1) and was identified using Arabidopsis aba2 mutants (CHENG et al., Plant Cell, 14, 2723-43, 2002; GONZALEZ-GUZMAN et al., Plant Cell, 14, 1833-46, 2002). Defects in two genes affect the last step of ABA biosynthesis (FIG. 1) one encoding the abscisic aldehyde oxidase apoprotein and the second an enzyme required for the sulfuration of the molybdenum cofactor. The aba3 mutant was shown to have a lesion in a gene encoding a molybdenum cofactor sulfurase (SCHWARTZ et al., Plant Physiol, 114, 161-6, 1997), and the corresponding gene was identified by positional cloning (BITTNER et al., J. Biol. Chem. 276, 40381-4, 2001; XIONG et al., Plant Cell 13, 2063-83, 2001), whereas the role of an aldehyde oxidase in the last step of ABA biosynthesis was confirmed, thanks to the Arabidopsis aldehyde oxidase3 (aao3) mutant (SEO et al., Proc Natl Acad Sci USA, 97, 12908-13, 2000).
Most of the Arabidopsis ABA-deficient mutants identified to date have been demonstrated to present germination that is resistant to paclobutrazol, or equivalent gibberellic acid (GA) biosynthesis inhibitors. This phenotype is related to the antagonistic effects of ABA and GA on germination making the balance between the levels of the two hormones determinant as to whether a seed germinates or not. In an ABA-deficient mutant the GA requirement for germination is thus less than in a wild-type seed and paclobutrazol has been used effectively as a screening method to identify mutants affected in ABA biosynthesis or signalling (JACOBSEN and OLSZEWSKI, Plant Cell, 5, 887-96, 1993; LEON-KLOOSTERZIEL et al., Plant J, 10, 655-61, 1996; NAMBARA et al., Plant Cell Physiol, 39, 853-8, 1998). In accordance with the role of ABA in responses to water deficit, ABA-deficient mutants also often show phenotypes related to defects in stomatal closure; increased water loss and a wilty phenotype on water deficit (SEO et al., Proc Natl Acad Sci USA, 97, 12908-13, 2000; KOORNNEEF et al., Theor. Appl. Genet. 61, 385-93, 1982; LEON-KLOOSTERZIEL et al., Plant Physiol, 110, 233-40, 1996). It has been shown that overexpression of Arabidopsis NCED genes, AtNCED3 and AtNCED6, increases endogenous ABA levels, and improves tolerance to water deficit (IUCHI et al., Plant J, 27, 325-33, 2001; LEFEBVRE et al., Plant J, 45, 309-319, 2006).